Table 1.
Echocardiographic and hemodynamic measures in the Sham and TAC groups.
Fig 1.
SIRT5 KO leads to increased AMP/ATP ratio and AMPK activation in HEK293T cells.
(A) Verification of SIRT5 KO in HEK293T cells by immunoblotting with the indicated antibodies. (B-C) Cytosolic and mitochondrial NADH levels were measured by genetically encoded fluorescent protein biosensors as described in ‘Materials and Methods’. (D-G) The AMP/ATP ratio is significantly increased in SIRT5 KO HEK293T cells. 1×106 cells were seeded into each well of six-well plates. After culture for 16, 48, and 72 hours, the cells were subjected to LC-MS/MS for metabolic profiling as described in ‘Materials and Methods’. Relative levels of ATP (D), ADP (E), AMP (F) and AMP/ATP ratio (G) were quantified. n = 3 or 4 for each cell line. Data are shown as mean ± SD of at least 3 independent experiments, two-tailed unpaired Student's t-test. *denotes the P < 0.05, **denotes the P < 0.01, and ***denotes the P < 0.001 for the indicated comparisons. n.s. = not significant. (H-I) AMPK activation in SIRT5 KO HEK293T cells. Cells were collected at the indicated culture periods, and AMPK T172 phosphorylation (H-I) and ACC S79 phosphorylation (I) were detected by immunoblotting using the indicated antibodies. WB bands for detecting the same proteins at 36, 48, 60, and 72 hours in (H) are from the same gels.
Fig 2.
Sirt5 KO leads to increased AMP/ATP ratio and AMPK activation in mouse hearts.
(A) Sirt5 deficiency suppresses ATP production in hearts of fasted mice. Female Sirt5 KO mice (n = 3) and WT littermates (n = 3) (4 weeks old, 16–19 g) were fasted overnight. Upon sacrifice, mouse hearts were harvested and then subjected to determination of ATP as described in ‘Materials and Methods’. (B-E) Sirt5 deficiency suppresses mitochondrial ATP production in mouse heart. Sirt5 KO mice (n = 7) and sex-matched WT control mice (n = 6) (16–28 weeks old) were fasted overnight. Upon sacrifice, mouse hearts were harvested for isolation of cardiac mitochondrion as described in ‘Materials and Methods’. Relative levels of ATP (B), ADP (C), and AMP (D) were measured by LC-MS/MS analysis, and the AMP/ATP ratio was calculated (E). (F) Sirt5 deficiency enhances AMPK activation in hearts of fasted mice. Sirt5 KO mice (n = 6) and sex-matched WT control mice (n = 6) (12 weeks old) were fed normally or fasted overnight. Upon sacrifice, mouse hearts were harvested, and AMPK T172 phosphorylation was detected by immunoblotting using the indicated antibodies. Data are shown as mean ± SD of at least 3 independent experiments, two-tailed unpaired Student's t-test. *denotes the P < 0.05 and ***denotes the P < 0.001 for the indicated comparisons. n.s. = not significant.
Fig 3.
Sirt5 KO induces increased lysine succinylation and decreased ATP synthase activity in mouse heart mitochondria.
(A-C) Lysine succinylation, malonylation and glutarylation are dramatically increased in mitochondria of Sirt5 KO mouse heart. Male Sirt5 KO mice (n = 3) and WT control mice (n = 3) (16–28 weeks old) were fasted overnight. Upon sacrifice, mouse hearts were harvested for isolation of cardiac mitochondria. Immunoblotting was performed using the anti-sucinyllysine antibody (A), anti-malonyllysine antibody (B), and anti-glutaryllysine antibody (C). Total protein loading was stained with Ponceau S. (D) Summary of previous proteomic studies identifying ATP synthase subunits as succinylation/malonylation/glutarylation substrates regulated by SIRT5. The numbers of lysine sites which are regulated by succinylation, malonylation, glutarylation and SIRT5 were listed. N.D. = not determined. (E-F) Sirt5 deficiency inhibits ATP synthase activity in hearts of fasted mice. Sirt5 KO mice (n = 9) and sex-matched WT control mice (n = 6) (16–28 weeks old) were fasted overnight. Upon sacrifice, mouse hearts were harvested for isolation of cardiac mitochondrion, and were then subjected to citrate synthase activity (E) and ATP synthase activity (F) assays as described in ‘Materials and Methods’. Data are shown as mean ± SD of at least 3 independent experiments, two-tailed unpaired Student's t-test. **denotes the P < 0.01 for the indicated comparison. n.s. = not significant.
Fig 4.
Sirt5 KO prevents left ventricular dilation in TAC mice.
(A) The HW/BW ratio between male Sirt5 KO mice and WT littermates at 5 weeks after TAC surgery (n = 6–10 per group). HW, heart weight; BW, body weight. (B-F) Left ventricular hypertrophic parameters between male Sirt5 KO mice and WT littermates at 5 weeks after TAC surgery (n = 6–10 per group), including left ventricular mass/body weight (LV mass/BW) ratio (B), left ventricular diastolic anterior wall thickness (LVAWTd) (C), left ventricular systolic anterior wall thickness (LVAWTs) (D), left ventricular diastolic posterior wall thickness (LVPWTd) (E), and left ventricular systolic posterior wall thickness (LVPWTs) (F). (G-J) Changes in other hallmarks of hypertrophic cardiomyopathy and cardiac fibrotic markers between male Sirt5 KO mice and WT littermates at 5 weeks after TAC surgery (n = 6–10 per group). Hypertrophic markers, Anp (G) and Bnp (H), and fibrotic markers, Collagen1a1 (I) and Collagen3a1 (J) mRNA expression in mouse hearts were determined by qRT-PCR. Data are shown as mean ± SD of at least 3 independent experiments, one-way ANOVA. *denotes the P< 0.05, **denotes the P < 0.01 and ***denotes the P < 0.001 for the indicated comparisons. n.s. = not significant.
Fig 5.
Sirt5 KO ameliorates cardiac dysfunction caused by TAC.
(A-F) Left ventricular cardiac functional parameters between male Sirt5 KO mice and WT littermates at 5 weeks after TAC surgery (n = 6–10 animals per group), including left ventricular internal diastolic diameter (LVIDd) (A), left ventricular internal systolic diameter (LVIDs) (B), left ventricular end-diastolic volume (LVEDV) (C), left ventricular end-systolic volume (LVESV) (D), left ventricular ejection fraction LVEF% (E), and left ventricular shortening fraction LVFS% (F). Data are shown as mean ± SD of at least 3 independent experiments, one-way ANOVA. *denotes the P< 0.05 and **denotes the P < 0.01 for the indicated comparisons. n.s. = not significant.
Fig 6.
Sirt5 KO promotes AMPK activation in hearts of TAC mice.
(A-D) Sirt5 deficiency suppresses ATP production in mouse hearts after TAC surgery. Relative levels of ATP (A), AMP (B), and ADP (C) in mouse hearts in both the Sham and TAC groups (n = 3 per group) were determined by LC-MS/MS analysis as described in ‘Materials and Methods’, and the AMP/ATP ratio was calculated accordingly (D). (E-H) Sirt5 KO promotes AMPK activation in mouse hearts after TAC surgery. Mouse heart samples were harvested as described above in (A-D), and AMPK T172 phosphorylation and ACC S79 phosphorylation were detected by immunoblotting using the indicated antibodies (E). Quantification of p-AMPK/AMPK ratio (F), p-ACC/ACC ratio (G), and p-4EBP1/4EBP1 ratio (H) were shown. Data are shown as mean ± SD of 3 independent experiments, one-way ANOVA. *denotes the P< 0.05, and **denotes the P < 0.01 for the indicated comparisons. n.s. = not significant.